This application is the U.S. National Stage entry under 35 U.S.C. xc2xa7371 of PCT/EP98/01973, filed Apr. 3, 1998.
The present invention concerns new sulphated compounds of hyaluronic acid and derivatives thereof having anticoagulant and antithrombotic activities and processes for their preparation. Said compounds are useful in the preparation of pharmaceutical compositions and biomaterials and in the production of coatings for biomedical objects.
Heparin is the sulphated glycosaminoglycan with the greatest biological activity. Its antithrombotic and anticoagulant properties are well known. Indeed, it is used in treatment for cardiovascular pathologies where there is a risk of thrombosis, and it has contributed notably to the successful outcome of open-heart surgery. The structure of heparin is not altogether known.
Commercial heparin comprises a range of 21 different kinds (Nader et al., 1974, Biochem. Biophys. Res. Commun. 57:488), with a molecular weight of between 3,000 and 37,500 Da, and with varying anticoagulant activity.
Heparin""s anticoagulant activity depends on its structural characteristics, for example, on the degree of sulphation, on the degree of dissociation, on the sequence of the COOxe2x80x94 and SO3 xe2x80x94 groups, on the shape and size of the molecule. These factors are important to the formation of the ion bonds responsible for heparin""s biological activity (Stivala et al., 1967, Arch. Biochem. Biophys. 122:40).
Because of the high density of its negative charge, heparin has a strong affinity for cations and its activity is pH-dependent. In particular, the N-sulphated group of its glucosamine residue plays a fundamental role in the interaction with the factors regulating the coagulative processes.
A significant reduction in the N-sulphated groups drastically reduces its anticoagulant and antithrombotic activities.
Many natural polysaccharides have been sulphated in order to obtain heparin-like products (Hoffman et al., 1982, Carbohydrate Res. 2:115; Kindness et al., 1980, Brit. J. Pharmac. 69:675; Horton et al., 1973, Carbohydrate Res. 30:349; Okada et al., 1979, Makromol. Chem. 180:813; Kikuchi et al., 1979, Nippon Kagaku Kaishi 1:127; Manzac et al., 1981, Proc. Third M.I.S.A.O. 5:504). Moreover, sulphuric, carboxy or sulphonated groups have been attached to synthetic polymers such as polystyrene (Kanmaugue et al., 1985, Biomaterials, 6:297) and polyurethanes (Ito et al., 1992, Biomaterials, 13:131).
However, the anticoagulant activity of these materials is much lower than that of heparin and depends on the type of substituent, the type of bond, the degree of substitution and the sequence.
Lastly, some chemical reactions for the sulphation of polysaccharides are known (WO 88/00211; EP 0340628; Carbohydrate Research, 158, 183-190, 1986) but no derivatives have ever been obtained which present, besides the chemical physical characteristics peculiar to polysaccharides, any new characteristics such as anticoagulant activity.
In the international patent application, Publication No. WO 95/25751, a process is described for the nonselective sulphation of hyaluronic acid and the derivatives thereof to obtain compounds with an antithrombotic activity.
Their ability to inactivate thrombin is due to the formation of electrostatic interactions depending on the charge density, which increases according to the degree of sulphation, while heparin""s activity is the consequence of a direct interaction with antithrombin III (T. W. Barrowcliffe et al., Journal of Pharmaceutical and Biomedical Analysis, Vol. 7, No. 2, pages 217-226, 1989; Peter D. J. Grootenhuiis et al., J. Am. Chem. Soc., 113, 2743-2747, 1991).
Heparin is widely used, although it does present side effects such as haemorrhagic effects, which prevent its being used too freely or without medical guidance.
Moreover, because of its chemical-physical characteristics, heparin cannot be used as a biomaterial but simply as a coating for other materials, and in sufficiently small quantities to avoid its causing localized bleeding.
Lastly, by acting directly on the coagulation factors, the anticoagulant action of heparin begins very rapidly and its duration, albeit dose-dependent, is generally similarly brief. These drawbacks limit its applicability in certain surgical fields such as cardiovascular surgery involving the implantation of devices requiring an absolute absence of thrombogenicity for a given length of time.
The present invention is directed to novel sulphated compounds of hyaluronic acid and the derivatives thereof, optionally salified, with an anticoagulant and antithrombotic activity, wherein the glucosamines are partially N-sulphated or partially N-sulphated and partially or totally O-sulphated in position 6, for the preparation of pharmaceutical formulations, biocompatible and bioabsorbable biomaterials with an anticoagulant activity and for the coating of biomedical objects and processes for their preparation.
The present invention also provides for a chemical process using a well-characterized starting product such as hyaluronic acid, which process allows for the selective sulphation of the amino group of glucosamine or the hydroxy group in the position 6, to thus obtain new sulphated derivatives of hyaluronic acid with an unaltered range of molecular weights and with an anticoagulant activity similar to that of heparin.
While the ability of hypersulphated polysaccharides to inactivate thrombin is due to the formation of electrostatic interactions depending on the charge density, which increases according to the degree of sulphation, the N-sulphated derivatives provided in the present invention appear to act on the coagulation factors by means of a specific mechanism similar to that of heparin.
Further advantages of the present invention are represented by the improved chemical-physical characteristics of the N-sulphated derivatives compared to those of the hypersulphated derivatives, making them suitable for the preparation of biomaterials for use in the fields of biomedicine and health care and in the pharmaceutical field.
Moreover, the possibility of obtaining a compound with an anticoagulant and non-thrombogenic activity by means of selective sulphation of just the amino groups and hydroxy groups in position 6, notably reduces the costs of the process compared to the preparation of hyaluronic acid with all the hydroxy groups homogeneously sulphated.
The term xe2x80x9cpartially 2-N-sulphated derivativexe2x80x9d of hyaluronic acid as used herein means a product obtained by means of a controlled sulphation reaction of the amino group of the glucosamine of hyaluronic acid, previously N-deacetylated according to the procedure described by P. Shaklee (1984) Biochem. J. 217, 187-197. The reaction proceeds as illustrated below; 
The term xe2x80x9cpartially 2-N-sulphated and 6-O-sulphated derivativesxe2x80x9d as used herein means the products of the chemical reaction illustrated in diagram 1, wherein, besides the amino group of glucosamine, the primary hydroxy function of the same residue is also totally or partially involved in the sulphation reaction, as illustrated below: 
The derivatives generated according to diagrams 1 and 2 can be used as intermediate reactants in the preparation of compounds, according to the procedure described in European patent 0216453 B1, wherein the carboxy function of the glucuronic residue of hyaluronic acid, partially 2-N-sulphated or partially 2-N-sulphated and partially or totally 6-O-sulphated, is partially or completely reacted with alcohols of the aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclic series, producing the respective partial or total esters: 
Moreover, it is possible to use the synthetic derivatives according to diagrams 1 and 2 as intermediates in the preparation of crosslinked compounds, according to the procedures described in European patents 0341745 B1 and 265116 B1 respectively, wherein a part or all of the carboxy groups belonging to the D-glucuronic residue are reacted: i) using condensing agents with the alcohol functions of the same polysaccharide chain or other chains, generating inner (or lactone) esters and intermolecular esters; ii) with poly-alcohols of the aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclic series, generating crosslinking by means of spacer chains.
The above-said sulphated compounds obtained according to the process of the present invention can be optionally salified with heavy metals, the heavy metals being selected from the group of metal elements in the 4th, 5th and 6th periods of the periodical table, such as silver, iron, cobalt, copper, zinc, arsenic, strontium, zirconium, antimonium, gold, cesium, tungsten, selenium, platinum, ruthenium, bismuth, tin, titanium, and mercury. These salts can be used in dermatology, ophthalmology, dentistry, stomatology, rheumatology, urology, gynecology, internal surgery, as food supplements and as anti-oxidant, antirheumatic, antitumoral, antiinflammatory, analgesic and anti-ulcer agents.
Lastly, the sulphated derivatives can be optionally salified with pharmacologically active substances such as antibiotics, antiinfective, antimicrobial, antiviral, cytostatic, antitumoral, antiinflammatory and wound healing agents, anesthetics, cholinergic or adrenergic agonists or antagonists, antithrombotic, anticoagulant, haemostatic, fibrinolytic and thrombolytic agents, proteins and their fragments, peptides, and polynucleotides,
Apart from their above noted antithrombotic and anticoagulant properties and their biocompatibility characteristics, the N-sulphated derivatives of the present invention can be used advantageously as antiviral agents, for example against human immune deficiency virus (HIV) and herpes, and as anti-inflammatories for systemic, topical or local use, for example by the rectal or vaginal routes both in the form of pharmaceutical compositions and in the form of biomaterials.
The present inventive compounds and their salts can therefore be used advantageously, either alone or in association with one another or with other pharmacologically active substances, in combination with a pharmaceutically acceptable carrier for the preparation of pharmaceutical compositions.
Such pharmaceutical compositions can be used, for example, as antithrombotic, anticoagulant, antiinflammatory, antiviral, anti-oedematous preparations, to accelerate wound healing, in the treatment of burns, sores, skin ulcers, dental decay, restenosis and infarction and to favor angiogenesis.
Of special interest are formulations and biomaterials for the transport and release of biologically active substances such as proteins and their fragments, peptides, polynucleotides, growth factors, enzymes, vaccines, substances used in the treatment of diseases associated with genetic defects such as those depending on enzymatic hypo- or hyperactivity due to defects of the gene encoding for a given enzyme, deforming diseases and hereditary disorders.
The sulphated derivatives according to the present invention can be associated with radioactive and non-radioactive substances used in contrast systems, and used as tracers in in vivo diagnostics in the identification and cure of tumoral or damaged tissues.
One considerable advantage is represented by the possibility of processing the sulphated compounds and their salts in various forms of biomaterials such as sponges, films, membranes, threads, tampons, nonwoven fabrics, microspheres, nanospheres, gauzes, gels, guide channels. These biomaterials, in one such form or several forms together, can be constituted by one or more sulphated derivatives and by their salts, optionally included in association with other natural, synthetic, semisynthetic polymers and, optionally, with biologically active substances.
Examples of the natural polymers which can be used are collagen, coprecipitates of collagen and glycosaminoglycans, cellulose, polysaccharides in the form of gels such as chitin, chitosan, pectin or pectic acid, agar, agarose, xanthan, gellan, alginic acid or alginates, polymannan or polyglycans, starch, natural gums. The semisynthetic polymers for example can be chosen from the group consisting of crosslinked collagen with agents such as aldehydes or precursors of the same, dicarboxylic acids or their halogenides, diamines, derivatives of cellulose, hyaluronic acid, chitin or chitosan, gellan, xanthan, pectin or pectic acid, polyglycans, polymannan, agar, agarose, natural gum and glycosaminoglycans. Lastly, examples of synthetic polymers that can be used are polylactic acid, polyglycolic acid or copolymers of the same or their derivatives, polydioxanes, polyphosphazenes, polysulphonic resins, polyurethanes, PTFE.
The biomaterials thus obtained can be used in the cardiovascular field or in all applications involving contact with the blood or with highly vascularized body tissues where the biomaterial used needs to be totally free of thrombogenicity, besides having characteristics of biocompatibility and biodegradability due to the use of the ester derivatives of hyaluronic acid.
The above noted biomaterials can be used to advantage in various fields of surgery: in internal surgery, osteoarticular surgery, surgery to the nerves, anastomosis, viscoelastic, ophthalmic, oncological, plastic, otorhinolaryngological, abdominal-pelvic, urogynaecological and cardiovascular surgery, such as in the preparation of cardiac valves, vascular stents, in the prevention of post-surgical adhesions, in the prevention of hypertrophic scarring.
Moreover, the sulphated compounds associated with fibrin, and possibly with other biologically active substances, can be used for the preparation of surgical glues.
The biomaterials according to the present invention can be used in other fields besides the surgical field, for instance in haemodialysis, cardiology, dermatology, ophthalmology, otorhinolaryngology, dentistry, gynaecology, urology, in extracorporeal blood circulation and oxygenation and in cosmetics.
The above noted biomaterials in their various forms can also be used to advantage as supports for cell cultures such as mesenchymal cells or mature cells to obtain connective tissue, glandular tissue and nerve tissue.
The instant biopolymers can also be used in processes to coat objects used both in the medical field and in other industrial sectors, to thereby give new biological characteristics to the surfaces of materials used as supports.
Examples of objects which can be thus coated are catheters, guide channels, probes, cardiac valves, soft tissue replacements, replacements of an animal origin such as cardiac valves from pigs, artificial tendons, bone and cardiovascular replacements, contact lenses, blood oxygenators, artificial kidneys, hearts, pancreas and liver, blood bags, syringes, surgical instruments, filtration systems, laboratory instruments, culture containers and containers for the regeneration of cells and tissues, supports for peptides, proteins, antibodies.
One method of coating the surfaces of these objects is the Plasma Coating technique described in international patent application No. WO96/24392, and illustrated in more detail hereafter in a preparation example. The process for the preparation of the compounds of the present invention mainly consists of two steps, the first involving the controlled N-deacetylation of the natural polysaccharide, and the second involving the specific sulphation reaction of the primary hydroxy or free amino functions of glucosamine.
Fractions of hyaluronic acid from biological and fermentation sources, with a molecular weight of between 5,000 and 5,000,000 Da, preferably between 50,000 Da and 300,000 Da, are solubilized in hydrazine hydroxide with a purity of no less than 98%, in a concentration range of between 1 and 50 mg/ml, preferably between 5 and 25 mg/ml. This solution is then supplemented with hydrazine sulphate in a weight/volume concentration varying between 0.1 and 3%, preferably 1%.
The reaction is conducted within a temperature range of 40 to 90xc2x0 C., preferably 60xc2x0 C., under agitation, for as long as it takes to reach the desired degree of N-deacetylation.
Table 1 hereafter reports the yield expressed as the percentage of free amino groups, in terms of time expressed as hours of reaction:
The reaction is then stopped by precipitation with a polar solvent, preferably ethanol. The precipitate is partially vacuum-dried and treated with a solution of iodic acid with a molarity range of between 0.1 and 1M, preferably 0.5M, and lastly, with iodohydric acid at a concentration of 57% (w/v). The pH of the solution is maintained between 5 and 7 by adding a solution of sodium acetate (10% w/v).
The aqueous phase containing the modified polysaccharide is extracted by repeated treatments with diethylether and then, once the yellow color has completely disappeared, the solution is treated again with ethanol.
The precipitate which forms, after further drying at 40xc2x0 C., is solubilized in water at a concentration of between 10 ng/ml and 40 ng/ml, preferably 25 ng/ml, and the solution is percolated through a column containing an ion exchange resin activated with a tetraalkyl-ammonium hydroxide, where the alkyl residue of the quaternary ammonium is constituted by a chain of between 1 and 4 carbon atoms; tetrabutylammonium hydroxide is preferably used.
The percolated product, represented by the quaternary ammonium salt of the modified polysaccharide, is then freeze-dried.
Method A
The quaternary ammonium salt, preferably of tetrabutyl-ammonium, of the partially N-deacetylated polysaccharide, is solubilized in an apolar solvent such as dimethyl sulphoxide, dimethyl formamide, dimethyl acetamide, N-methyl-pyrrolidone, preferably dimethyl formamide (DMFA), at a concentration of between 5 and 50 mg/ml (preferably 25 mg/ml).
The organic solution is supplemented with another solution obtained by solubilizing the sulphating complex constituted by dimethylformamide sulphotrioxide (DMFA-SO3), in DMFA, at a concentration varying between 50 and 200 mg/ml and preferably 100 mg/ml. The quantity of complex to be used, expressed in moles of SO3, proves surprisingly to be equivalent to the moles of amino groups released by the N-deacetylation reaction.
The sulphation reaction proceeds at a temperature of between 0xc2x0 and 20xc2x0 C., preferably 4xc2x0 C. for no longer than 4 hours and is then stopped by adding cold, distilled water.
The reaction solvent is first purified by precipitating the partially 2-N-sulphated hyaluronic acid with ethanol and then dialysing the resolubilized product with distilled water.
Lastly, the solution is freeze-dried and the solid product thus obtained undergoes chemical-analytical characterization to determine the degree of N-sulphation and the mean molecular weight (Table 2).
Method B
The quaternary ammonium salt, preferably of tetrabutyl-ammonium, of the partially N-deacetylated polysaccharide is solubilized in an apolar solvent such as dimethylsulphoxide, demethylformamide, dimethylacetamide, N-methylpyrrolidone, preferably dimethylformamide (DMFA), at a concentration of between 5 and 50 mg/ml, preferably 30 mg/ml.
The organic solution is supplemented with another solution obtained by solubilizing the sulphating complex constituted by dimethylformamide sulphotrioxide (DMFA-SO3), in DMFA, at concentrations varying between 50 and 200 mg/ml and preferably 100 mg/ml. The quantity of complex used, expressed as moles of SO3, prove surprisingly to be equivalent to the moles of amino groups released by the N-deacetylation reaction.
The sulphation reaction proceeds at a temperature of between 0xc2x0 and 20xc2x0 C., preferably at 4xc2x0 C. for 4 hours. A solution prepared by solubilizing the pyridine-sulphotrioxide complex in dimethylsulphoxide in such a quantity that the ratio between the moles of SO3 of the sulphating agent and the moles of xe2x80x94CH2OH comes between 1.1 and 1.3. Larger quantities of reagent may favor any substitution reactions in other alcohol groups (secondary) of the polysaccharide chain.
The reaction then proceeds for another 16 hours at least after which it is stopped by adding cold, distilled water.
All subsequent steps concerning the purification of the modified polysaccharide are those described in xe2x80x9cmethod Axe2x80x9d.
The analytical characterization performed on the derivatives obtained confirmed that the sulphation method proves surprisingly not only to substitute all the amino groups obtained by the partial N-deacetylation, but also results in the complete substitution of the primary alcohol group of the glucosamine residue of hyaluronic acid (Table 3).
Moreover, by varying the molar quantities of the pyridine-SO3 complex according to the primary hydroxyl groups (molar ratio of between 0.1 and 1), xe2x80x9cmethod Bxe2x80x9d enables a series of partially 2-N-sulphated and partially 6-O-sulphated derivatives to be obtained.
The compounds prepared as described previously are characterized from a biological point of view in such a way as to determine their anticoagulant activity. As reference products, we used the sulphated derivatives of hyaluronic acid obtained according to the method described in international patent application No. WP 95/25751, where the primary and secondary hydroxy groups of the polymer chain are substituted with xe2x80x94SO3xe2x80x94groups according to the quantity of sulphating agent that is used. Moreover, unfractionated heparin (UF heparin) is used as a further reference product, the efficacy of which as an anticoagulant drug has been widely acknowledged for many decades.
Subsequently, the thrombin time (TT test) and whole blood coagulation time (WBCT) were assessed for the following compounds:
The ability of the N-sulphated derivatives to prolong blood coagulation time is measured by the thrombin time test performed with a coagulometer. The time it takes to transform fibrinogen into fibrin after the addition of thrombin to a blood sample is determined in the presence of the polymer used as starting material. The test loses its significance when the time exceeds 120 seconds. The coagulation time is determined by simply observing the time it takes for a sample of human blood to coagulate in the presence of the test material. Any times exceeding two hours are not considered.
Surprisingly, it seems clear that the chemical modification involving the amino group of the glucosamine residue of the polysaccharide chain induces a notable effect on the coagulation time. An identical result can only be obtained by drastically modifying the chemical structure of HA, that is, by substituting all the hydroxyls available per monomeric unit (four) with as many sulphonic groups.
The data obtained show that a degree of sulphation of 0.25, deriving from the previous N-deacetylation and subsequent N-sulphation, is sufficient to act specifically with the factors involving the terminal part of the coagulation cascade, thereby inhibiting the process of transformation of the fibrinogen into fibrin (anticoagulant activity). The presence of the sulphur group in position 6-O of the glucosamine residue does not seem to be a determining factor in the performance of this activity. However, in association with 2-N-sulphated groups, it may play a fundamental role in the inhibition of the Xa factor by interaction with ATIII (antithrombotic activity) and in the inhibition of the factor VIIIa (or vWf) in regulating platelet activation and proliferation.
The chemical-analytical profiles of all the derivatives prepared and described in the present invention have been characterized. The following analytical methodologies were used:
Degree of sulphation (% of sulphur): after undergoing complete combustion in an oxygen-rich environment, the product was analyzed by HPLC using an ionic chromatography technique.
Percentage of 2-N-sulphation: this parameter is indirectly determined by measuring the total sulphonic groups present both before and after N-desulphation of the product, using the method described by Nagasawa (Carbohy. Res. 1977, 58, pages 47-55). The difference between the two values represents the quantity of SO3-groups linked to the amino group of glucosamine.
Percentage of N-deacetylation: this parameter is determined by the method described by J. Riesenfeld (Analy. Bioch. 1990, vol. 188, pages 383-389).
Mean molecular weight: this parameter is determined by GPC, using a set of Shadex and B-803 and B-806 columns, a multi-angle-laserlight-scattering monitor (MALLS) and a refractometer to measure the index of refraction (RI).